Mutations in rpoB That Confer Rifampicin Resistance Can Alter Levels of Peptidoglycan Precursors and Affect β-Lactam Susceptibility

Abstract

Bacteria can adapt to stressful conditions through mutations affecting the RNA polymerase core subunits that lead to beneficial changes in transcription. In response to selection with rifampicin (RIF), mutations arise in the RIF resistance-determining region (RRDR) of rpoB that reduce antibiotic binding. These changes can also alter transcription and thereby have pleiotropic effects on bacterial fitness. Here, we studied the evolution of resistance in Bacillus subtilis to the synergistic combination of RIF and the β-lactam cefuroxime (CEF). Two independent evolution experiments led to the recovery of a single rpoB allele (S487L) that was able to confer resistance to RIF and CEF through a single mutation. Two other common RRDR mutations made the cells 32 times more sensitive to CEF (H482Y) or led to only modest CEF resistance (Q469R). The diverse effects of these three mutations on CEF resistance are correlated with differences in the expression of peptidoglycan (PG) synthesis genes and in the levels of two metabolites crucial in regulating PG synthesis, glucosamine-6-phosphate (GlcN-6-P) and UDP-N-acetylglucosamine (UDP-GlcNAc). We conclude that RRDR mutations can have widely varying effects on pathways important for cell wall biosynthesis, and this may restrict the spectrum of mutations that arise during combination therapy. IMPORTANCE Rifampicin (RIF) is one of the most valued drugs in the treatment of tuberculosis. TB treatment relies on a combination therapy and for multidrug-resistant strains may include β-lactams. Mutations in rpoB present a common route for emergence of resistance to RIF. In this study, using B. subtilis as a model, we evaluate the emergence of resistance for the synergistic combination of RIF and the β-lactam cefuroxime (CEF). One clinically relevant rpoB mutation conferred resistance to both RIF and CEF, whereas one other increased CEF sensitivity. We were able to link these CEF sensitivity phenotypes to accumulation of UDP-N-acetylglucosamine (UDP-GlcNAc), which feedback regulates GlmS activity and thereby peptidoglycan synthesis. Further, we found that higher CEF concentrations precluded the emergence of high RIF resistance. Collectively, these results suggest that multidrug treatment regimens may limit the available pathways for the evolution of antibiotic resistance.

Keywords: Bacillus subtilis; Mycobacterium tuberculosis; RNA polymerases; antibiotic resistance; antibiotic synergy; metabolomics; peptidoglycan; rifampicin; β-lactams.

FIG 1

(A and B) Synergy between rifampicin (RIF) and cefuroxime (CEF) monitored by growth kinetics. Cell density was monitored after treatment with sub-MIC levels of CEF alone (A) or in the presence of 0.06 μg/mL RIF (B). The observed lag phases were all less than 5 h with CEF alone and increased to nearly 15 h with the combination treatment, as highlighted by the dashed lines.

FIG 2

Evolution of WT B. subtilis to achieve RIF and CEF resistance. (A) Schematic of the evolution experiment performed in the presence of the RIF + CEF combination; WGS, whole-genome sequencing. (B and C) RIF (B) and CEF (C) susceptibilities as measured by zone of inhibition for the 10 passages evolved under the 3 combination treatments. The three RIF and CEF concentrations used for evolving the cells are mentioned in the legend. The control group consists of 10 passages of WT cells that have not been treated with any drugs. The shaded bars in C represent the zone of lower density.

FIG 3

Drug susceptibilities of rpoB mutants. (A) Zone of inhibition against RIF and CEF for different rpoB mutants (note that only P520L had a detectable inhibition zone with RIF). (B) Zone of inhibition for β-lactams oxacillin, ampicillin, and penicillin and other cell wall-inhibiting drugs, such as nisin, vancomycin, and fosfomycin for the common clinically associated RIF-resistant rpoB mutants. Significance was defined as a P value of <0.001. A comparison was done between all mutants treated with the same drug. No comparison was done between drugs. A strain with a significant difference compared to others is given a different letter, and “a,b” indicates a value that is not significantly different from those strains in either group a or group b.

FIG 4

The effect of rpoB mutations on peptidoglycan (PG) synthesis. (A) Schematic of the PG synthesis pathway. (B and C) Expression levels of enzymes (B) and regulators (C) involved in PG synthesis in WT and rpoB mutants S487L, H482Y, and Q469R as determined by real-time PCR. The expression levels were calculated by the 2^−ΔCT method. gyrA was used as the internal control to normalize the levels of the genes of interest. The values are plotted on a log₁₀ scale. Significance was calculated by two-way ANOVA with Tukey’s multiple-comparison test. The two asterisks (**) indicate P values less than 0.001.

FIG 5

Metabolite levels in rpoB mutants. (A) Metabolite levels of pyruvate, which indicate the flux through glycolysis. (B) Metabolite levels of GlcN-6-P, which determine flux into PG synthesis. (C) Metabolite levels of UDP-GlcNAc, which indicate the rate of PG formation. Same letters define a data set with no significant difference. Significance was defined as a P value of <0.0001. A comparison was performed between all strains for each metabolite. A strain with a significant difference compared to others is given a different letter.

FIG 6

The importance of GlmR activity in CEF sensitivity. The sensitivity of WT and rpoB mutants with and without the deletion of glmR as measured by zone of inhibition. An asterisk (*) indicates P values less than 0.0001; ns, not significant.

FIG 7

Perturbation of GlcN-6-P and UDP-GlcNAc levels in cells. (A and B) The sensitivity of WT and rpoB mutants against CEF as measured by zone of inhibition on medium supplemented with 20 mM GlcNAc and on deletion of gamA, which directs GlcN-6-P toward glycolysis (A), and after induction of the phosphoglucosamine mutase glmM and pgcA* and phosphoglucomutase pgcA (B). An asterisk (*) indicates P values less than 0.01.

FIG 8

Interpretation of experimental perturbations predicted to affect UDP-GlcNAc levels. The text and arrow in blue summarize the data for the CEF^R S487L mutant. This mutant has low levels of UDP-GlcNAc. Thus, GlmR is free to stimulate GlmS activity, and cells are predicted to maintain a high rate of PG synthesis detected by high levels of GlcN-6-P. The text and arrow in red summarize the data for the CEF^S H482Y mutant. This mutant has high levels of UDP-GlcNAc, which would bind with GlmR. The bound GlmR is unavailable to stimulate GlmS activity and is thereby predicted to reduce PG synthesis. Three perturbation scenarios are also presented. In green, cells were supplemented with 20 mM GlcNAc or gamA was deleted. Both led to higher flux toward PG synthesis independent of GlmS, thereby bypassing the bottleneck in the H482Y mutant and leading to elevated CEF resistance. In orange, induction of glmM or pgcA* is predicted to increase the levels of UDP-GlcNAc but only in S487L, which has high levels of GlcN-6-P. Thus, this treatment is predicted to block GlmR-dependent GlmS activation in S487L, reduce PG synthesis, and thereby contribute to CEF sensitivity. These inferences are supported by analysis of the effects of a glmR deletion (purple). In S487L, we observed low UDP-GlcNAc levels and predict that GlmR is activating GlmS. Consistently, deletion of glmR makes the S487L strain more CEF sensitive. In contrast, in H482Y, we predict that the high observed UDP-GlcNAc levels will keep GlmR sequestered in an inactive state, and consistently there is no effect of deleting glmR.